Polyunsaturated fatty acids are of major importance in animal health as they have roles in the maintenance of membrane structure and function, in the regulation of cholesterol synthesis and transport, in the prevention of water loss from the skin, and as precursors of eicosanoids, including prostaglandins and leucotrienes. In animals, members of this class of fatty acids are synthesized from the essential fatty acid linoleic acid (C18:2Δ9,12), the first step being the desaturation to gamma-linolenic acid (GLA; C18:3Δ6,9,12) catalyzed by delta-6 desaturase. Clinical trials have shown that dietary supplementation of GLA may be effective in treating a number of ailments (e.g., atopic eczema, mastalgia, diabetic neuropathy, viral infections, and some types of cancer) (Jiang et al. (1998) Crit. Rev. Oncol. Hematol. 27:179-209; Kruger, and Horrobin (1997) Prog. Lipid Res. 36:131-51). Oils containing GLA are therefore widely used as a general health supplement and have been registered for pharmaceutical use.
In the plant kingdom, GLA is an uncommon fatty acid. Major commercial sources of GLA are evening primroses (Oenothera spp.), in which GLA accounts for about 8 to 10% of the seed oil, and borage (starflower: Borago officinalis) wherein seeds contain some 20 to 25% GLA. These plants, however, suffer from poor agronomic performance and low yield. There is therefore considerable interest in increasing the GLA content of existing crops and in producing GLA in a conventional oil crop. Expression of a cDNA encoding the delta-6 fatty acid desaturase from developing seeds of borage in transgenic tobacco plants resulted in accumulation of GLA and octadecatetraenoic acid (C18:4 Δ6,9,12,15) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage delta-6 fatty acid desaturase differs from other previously characterized higher plant desaturase enzymes by the presence of an N-terminal domain related to cytochrome b5 (Sayanova et al. (1997) Proc. Natl. Acad Sci USA 94:4211-4216). This desaturase does not appear to have an N-terminal cleavable endoplasmic reticulum-targeting signal, but the hydrophobic regions present in the protein would be sufficient to allow it to associate with the endomembrane system. A tripartite motif containing eight conserved histidines has been identified in almost all membrane desatuases (HX(3-4)HX(7-4)HX(2-3)HHX(61-189)HX(2-3)HH). In Anabaena and borage delta-6 desaturase a glutamine residue replaces the first histidine of the third element.
Membrane and reserve lipids of plants contain fatty acids with different degrees of unsaturation which are controlled by different desaturase enzymes. A cDNA isolated from ripening sunflower embryos encodes a protein with the conserved three histidine domains characteristic of membrane-bound desaturases. This cDNA also encodes a fusion protein composed of an N-terminal cytochrome b5 and a domain similar to membrane-bound acyl lipid desaturases (Sperling et al. (1995) Eur. J. Biochem. 232:798-805). Expression of homologous cDNAs from Brassica napus and Arabidopsis thaliana in Saccharomyces cerevisiae results in significant proportions of new Δ8,9-cis/trans-phytosphingenines that accompany the residual C18-phytosphinganine predominating in wild-type yeast cells. These genes encode new members of the cytochrome b5 superfamily which function as a stereounselective sphingolipid desaturase and show trans-activity (Sperling et al. (1998) J. Biol. Chem. 273: 28590-28596).
The enzymes encoded by the picramnia cDNAs included in this application possibly catalyze the formation of a triple bond since picramnia seeds accumulate large amounts of tariric acid (18:1 delta-6-acetylenic), an unusual fatty acid that has a triple bond (or acetylenic bond) at the delta-6 carbon. Tariric acid has many of the same industrial uses ascribed to petroselenic acid, thus these cDNAs should be useful in the production of novel fatty acids in the seed oils of transgenic plants. Although with similarities to the delta-6 desaturase, the enzymes encoded by the corn, soybean, and wheat sequences described herein are sphingolipid desaturases since these plants do not produce delta 6 double bonds like picramnia and borage.